Priming of phosphatidic acid production by staurosporine in f-Met-Leu-Phe-stimulated human neutrophils--correlation with respiratory burst.
Staurosporine, a microbial alkaloid known as a potent though non specific PKC inhibitor, enhances the production of superoxide anion (respiratory burst) of human polymorphonuclear leukocytes (PMN) stimulated by chemoattractants such as f-Met-Leu-Phe (fMLP). To gain insights into the mechanisms of this priming, we analysed staurosporine effects on formation of second messengers issued from phospholipase D (PLD), i.e., phosphatidic acid (PA) and its dephosphorylated form, diglycerides (DG). PA and DG were measured by two methods, in mass and after the labelling of PMN with a phosphatidylcholine precursor, [3H]-1-O-alkyl-2-lyso-3-phosphatidylcholine. Treatment of labelled PMN with low concentrations of staurosporine (12.5 and 50 nM) which prime respiratory burst had no significant effect on basal amounts of tritiated PA and DG, but potentiated fMLP-mediated formation of [3H]PA and phosphatidylethanol (PEt) pointing to a priming of PLD activity. PA mass in resting PMN increased (approximately 80 +/- 7%) in the presence of high drug concentrations only (250-500 nM), with no change in basal DAG mass. Low staurosporine concentrations (6.25-25 nM) markedly potentiated PA mass formation induced by fMLP and positive correlation (R = 0.95) was found between enhanced superoxide formation and generation of PA but not DG. Furthermore, cytochalasin B, which is known to prime PA production induced by fMLP, synergised the priming of respiratory burst by staurosporine, which further suggests a functional role of PA. In contrast to staurosporine, the more selective PKC inhibitor GF109203X neither stimulated PLD nor primed fMLP-induced PLD or respiratory burst. These data indicate that priming of fMLP-mediated PMN respiratory burst by staurosporine correlates with PA formation. This priming may be linked to alteration of early signalling events upstream of PLD rather than to feedback inhibition of PKC.
Rais
S
S
Laboratoire d'Hématologie, INSERM U294, Hôpital Bichat, Paris, France.
Pédruzzi
E
E
Dang
M C
MC
Giroud
J P
JP
Hakim
J
J
Périanin
A
A
D004075:Diglycerides / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020404:Glycerophospholipids; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D010711:Phosphatidate Phosphatase / Q000378:metabolism; D010712:Phosphatidic Acids / Q000096:biosynthesis* / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D016897:Respiratory Burst / Q000187:drug effects; D019311:Staurosporine / Q000494:pharmacology*
D004075:Diglycerides; D004791:Enzyme Inhibitors; D020404:Glycerophospholipids; D007211:Indoles; D008301:Maleimides; D010712:Phosphatidic Acids; C051521:phosphatidylethanol; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D010711:Phosphatidate Phosphatase; D010739:Phospholipase D; D019311:Staurosporine; C070515:bisindolylmaleimide I